藤黄微球菌Rpf结构域及其突变体基因的克

发布时间：2018-01-05 00:21

本文关键词：藤黄微球菌Rpf结构域及其突变体基因的克隆、表达及生物活性的初步研究　出处：《第四军医大学》2008年硕士论文　论文类型：学位论文

【摘要】： 结核病(Tuberculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis,MTB)所致的、以呼吸系统感染为主的传染病。近年来,由于卡介苗(BCG)保护性的不完善、耐药MTB菌株的出现、艾滋病的流行以及人口流动等因素的增加,使得TB的发病率呈日益上升的趋势,再次成为危害人类健康的世界性疾病。BCG是目前预防TB的唯一疫苗,但其不能有效预防成人TB,因此,积极研究TB的致病及免疫机制,对于开发更为有效的诊断、预防和治疗TB的新技术具有重要意义。藤黄微球菌(M.luteus)中的复活促进因子(Resuscitation-promoting factor,Rpf)是第一个被发现的细菌生长因子,它具有促进休眠菌的复苏以及促进细菌快速生长的作用。序列对比分析发现,在其它的一些革兰氏阳性细菌如MTB、麻风分枝杆菌、蓝色链霉菌、谷氨酸棒杆菌等中也存在30多个与Rpf同源的蛋白。研究发现MTB基因组可编码5种Rpf样蛋白,分别为Rv0867C(Rpf A)、Rv1009(Rpf B)、Rv1884C(Rpf C)、Rv2389C(Rpf D)和Rv2450C(Rpf E),推测这些蛋白也具有促进MTB快速生长的作用。此外,这些蛋白均具有Rpf样结构域,即高度保守的70aa残基,该保守区域对Rpf的复苏功能是至关重要的,单独的Rpf结构域具有与Rpf蛋白一致的生物学功能。进一步的研究发现,Rpf结构域中都有一个高度保守的谷氨酸残基(glu54),为Rpf活性所必需,与溶菌酶催化活性相符。实验目的: 利用大肠杆菌表达系统,表达并纯化出M. luteus Rpf结构域及其突变体蛋白,并进一步研究其生物学活性。实验方法和结果: 1. Rpf结构域及其突变体蛋白的表达及纯化采用聚合酶链反应(PCR)从M. luteus基因组中扩增出Rpf结构域及其E54A和E54K两种突变体基因,用限制性内切酶消化后克隆入pMD-18T载体中,经测序证实,与GenBank公布的序列完全相同,突变位点与设定一致。将测序正确的基因分别亚克隆到pGEX-4T-1表达载体中,酶切鉴定阳性重组质粒,转化大肠杆菌,并用IPTG进行诱导表达。SDS-PAGE分析表明成功地表达了Rpf结构域及其突变体融合蛋白,用Rpf结构域单克隆抗体(mAb)进行Western-blot分析证实,其大小为32kD,与预计的融合蛋白大小相符。用GS-4B亲和色谱柱纯化获得了目的蛋白。 2. Rpf结构域及其突变体融合蛋白生物学活性的检测将耻垢分枝杆菌(Mycobacterium smegmatis,M.S)休眠菌以1:150的比例稀释。把各种融合蛋白按照不同的浓度加入到M.S休眠菌中,并分别用相应的抗体进行干预。37℃振荡培养,每4 h取少量培养液测A600nm值,根据测定结果绘制生长曲线图。结果表明:Rpf结构域蛋白对M.S具有促进复苏和促生长的作用,使M.S的培养周期缩短。而且Rpf结构域蛋白具有和Rpf蛋白相同的生物学活性。E54K突变后的Rpf蛋白对于M.S的生长有一定的抑制作用,而E54A无明显的抑制生长作用。结论: 获得了Rpf结构域及其两种突变体蛋白E54K和E54A,Rpf结构域蛋白与完整的Rpf蛋白一样对M.S具有一定的复苏和促生长作用;而加入Rpf及Rpf结构域抗体后,其作用被抵消,使M.S维持正常生长。E54K突变体对于休眠的M.S的生长有一定的抑制作用。E54A对于休眠的M.S无明显的抑制作用。这些结果为研究Rpf结构域突变体在抑制M. luteus的生长,进而研究其对MTB生长的影响,以及Rpf结构域和其突变体抗体在TB预防及临床快速诊断中的作用奠定基础。
[Abstract]:Tuberculosis (Tuberculosis, TB) by Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) caused by, on respiratory system infection of infectious diseases. In recent years, due to BCG (BCG) protection is not perfect, the emergence of drug-resistant MTB strains, and to increase the prevalence of HIV / AIDS population movements and other factors, makes the TB the incidence is increasing, harm to human health worldwide disease.BCG is currently the only vaccine against TB become again, but it can not effectively prevent adult TB, therefore, study on the pathogenesis and immunity of TB positive, for the development of more effective diagnosis, prevention and treatment of TB new technology has important significance.
Micrococcus luteus (M.luteus) in the resuscitation promoting factor (Resuscitation-promoting factor, Rpf) is the first discovered bacterial growth factor, it can promote the recovery and promote the role of dormant bacteria, bacteria grow fast. Sequence analysis found that in some other gram positive bacteria such as MTB, Mycobacterium leprae, Streptomyces coelicolor more than 30, homologous with Rpf protein also exists in Corynebacterium glutamicum. Research found that MTB genome encoding 5 Rpf proteins, respectively Rv0867C (Rpf A), Rv1009 (Rpf B), Rv1884C (Rpf C), Rv2389C (Rpf D) and Rv2450C (Rpf E), suggested that these proteins MTB has a role in promoting rapid growth. In addition, these proteins are Rpf like domain that is highly conserved 70AA residues, conserved region of Rpf is crucial to the recovery of the function of the single Rpf domain is consistent with the biology of Rpf protein Further studies revealed that a highly conserved glutamic acid residue (glu54) in the Rpf domain is necessary for Rpf activity, which is consistent with lysozyme catalytic activity.
Objective:
The M. luteus Rpf domain and its mutant protein were expressed and purified by the Escherichia coli expression system, and its biological activity was further studied.
Experimental methods and results:
Expression and purification of 1. Rpf domain and its mutant protein
Polymerase chain reaction (PCR) amplified Rpf domain and its E54A and E54K two kinds of mutant gene from M. luteus genome, with restriction endonuclease digestion and cloned into pMD-18T vector and confirmed by sequencing, identical with the published sequences of GenBank mutation and set. Then the correct sequence of the gene were sub cloned into pGEX-4T-1 expression vector, enzyme digestion of recombinant plasmid was transformed into Escherichia coli, and induced expression of.SDS-PAGE analysis showed that the successful expression of the Rpf domain and its mutants with IPTG fusion protein, Rpf domain monoclonal antibody (mAb) Western-blot analysis confirmed that its size is 32kD, with the expected protein with the size of fusion. Purified the fusion protein by GS-4B affinity.
Detection of biological activity of 2. Rpf domain and its mutant fusion protein
The Mycobacterium smegmatis (Mycobacterium smegmatis M.S) dormant bacteria in 1:150 dilution ratio. The fusion protein with different concentrations of M.S added to the dormant bacteria, respectively and the corresponding antibody intervention.37 C shaking culture, every 4 h to take a small amount of medium A600nm value, according to the determination results of growth curve figure. The results show that the Rpf domain protein can promote the recovery and growth promoting effect on M.S, the M.S and shorten the training cycle. Rpf domain protein has the same biological activity of.E54K and Rpf protein mutation Rpf protein has certain inhibitory effect on the growth of M.S, while E54A had no obvious growth inhibition.
Conclusion:
The Rpf domain and its two mutants E54K and E54A, Rpf domain and complete Rpf protein of M.S has a certain recovery and growth promoting effect; and adding Rpf and Rpf domain antibodies, its role is to offset, M.S to maintain normal growth of.E54K mutant for M.S with dormancy the inhibitory effect of.E54A for dormant M.S had no obvious inhibitory effect. These results for the study of Rpf domain mutants in M. suppressed the growth of luteus, and to study its effects on the growth of MTB, lay the foundation and function of Rpf domain and its mutant antibody in TB prevention and clinical rapid diagnosis.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378

【引证文献】